Profiling Drosophila third instar larval eye discs with disruptions in Hippo and Rbf pathways by 10X single-cell RNA-Sequencing

We performed single-cell RNA-sequencing experiments to understand how the Rbf and Hippo pathways collaborate to maintain cell identity. Drosophila melanogaster photoreceptor neurons with dual mutation in Rbf[120a] and wts[x1] specify normally before abruptly dedifferentiating. We performed scRNA-Seq on w1118, Rbf[120a] single mutant, wts[x1] single mutant, and Rbf[120a] wts[x1] double mutant tissues to better understand the dedifferentiation phenotype. We identified transcriptional changes that were unique to double mutant tissue. We then validated these experiments using a novel dedifferentiation model expressing constitutively active yki[S111A,S168A,S250A] in Rbf[120a] mutant tissue using the GMR-GAL4 driver. We performed single-cell experiments on eye tissues expressing yki[S111A,S168A,S250A] in Rbf wildtype and mutant backgrounds, as well as tissues co-expressing Hth and yki. We then analyzed these experiments to determine the dedifferentiation transcriptional program. 10X single-cell RNA-sequencing on single cell suspensions from third instar larval eye discs dissected at the optic stalk and eye antennal border. In total 2 wildtype w1118, 2 Rbf[120a] mutant, 1 wts[x1] mutant, 1 Rbf[120a] wts[x1] double mutant, 1 GMR-GAL4>ykiS111A,S168A,S250A, 1 Rbf[120a];GMR>yki[S111A,S168A,S250A], and 1 GMR>Hth, yki[S111A,S168A,S250A], replicates were included to explore the role of the RB and Hippo pathways in maintaining photoreceptor specification.